JP4543188B2 - RNA sequences that act as RNAi for human thymidylate synthase - Google Patents

Description

  The present invention relates to compositions for the treatment and prevention of cell proliferative diseases.

  5-FU, UFT, CDDP, TS1, and the like targeting thymidylate synthase (TS) are known as main anticancer agents at present. Thymidylate synthase is an enzyme required for DNA synthesis and is expressed more in tumors with higher malignancy. However, it has been found that even the above-mentioned anticancer agents, which should theoretically suppress this enzyme, are effective only for a limited number of specific cancer types. The reason is that many malignant tumors express a large amount of an enzyme called DPYD that metabolizes these anticancer drugs, so that the anticancer drugs are metabolized in cancer cells and the effects are not achieved. it is conceivable that.

  For example, it has been found that most of the malignant tumors of the hepatobiliary system do not effectively act due to DPYD activity. Recently, anticancer drugs called UFT and TS1 were developed. These are combined with uracil, gimeracil and the like for suppressing DPYD, but even these cannot be said to be sufficient as an anticancer agent. Furthermore, these drugs have the disadvantage that they have strong side effects and are expensive.

  However, the anti-cancer drugs targeting TS are not sufficient, but show some effects. Therefore, if an anti-cancer drug targeting TS that is not affected by DPYD activity is developed, there is currently no suitable anti-cancer drug. It is thought that it can be an effective anticancer agent for some cancer types.

  In view of this, the present inventors have focused on the RNA interference (RNAi) method, which has been attracting attention as a method capable of suppressing the expression of a specific gene in recent years. RNAi suppresses specific gene expression by double-stranded RNA degrading mRNA having its sequence specificity or suppressing translation of the mRNA. Therefore, the knockdown method using RNAi has been attracting attention as a technique capable of suppressing specific gene expression easily and inexpensively, for example, in application to gene therapy.

Nat. Rev. Cancer, 2003 May; 3 (5): 330-338

  Develop TS inhibitors that can be effective anticancer agents for a wide variety of cancers in humans.

  An RNAi molecule targeting human TS mRNA for inhibiting human TS protein production is provided.

  Thereby, it is possible to provide an anticancer agent that is not affected by the expression level of DPYD and has a universal and sufficient effect on various human malignant tumors. For example, biliary malignant tumors are extremely difficult to treat because there is no sufficiently effective anticancer drug due to high expression of DPYD, and the prognosis of surgical therapy is extremely poor. Anticancer agents are very likely to be effective against these cancers. Therefore, an anticancer agent having no or few side effects is provided.

Accordingly, the present invention provides the following aspects:
1. The following (a) and (b):
(A) an RNA comprising a sequence consisting of at least 19 consecutive nucleotides in the sequence shown in any one of SEQ ID NOs: 1 to 4;
(B) an RNA having a sequence that hybridizes to the RNA of (a) under stringent conditions;
Is a hybridized double-stranded RNA, which can act as an RNAi molecule for targeting human thymidylate synthase (TS) and reducing the expression level of the enzyme in the cell;
2. The following (a) and (b):
(A) an RNA comprising a sequence consisting of at least 19 consecutive nucleotides in any one of the sequences shown in SEQ ID NOs: 1 to 4;
(B) an RNA having a sequence that hybridizes to the RNA of (a) under stringent conditions;
Are connected in tandem, and the connected part is
(C) RNA having one sequence selected from the group consisting of CUUCCUGUCA (SEQ ID NO: 27), UUCAAGAGA, CCACC, CUCGAG, CCACACC, UUCAAGAGA, AUG, CCC and UUCG;
(C) wherein the portion (c) has a loop structure when the above (a) and (b) are hybridized to form a double strand, human thymidyl RNA that can act as an RNAi molecule for targeting acid synthase (TS) and reducing the expression level of the enzyme in cells;
3. The RNA according to 1 or 2 above, wherein the sequence of the RNA of (b) is completely complementary to the sequence of (a);
4). The RNA according to any one of 1 to 3 above, wherein (a) is an RNA having the sequence shown in any one of SEQ ID NOs: 1 to 4;
5). The RNA according to 2 above, which has the sequence shown in any one of SEQ ID NOs: 5 to 8;
6). DNA encoding the RNA according to any one of 1 to 5 above;
7). DNA of said 6 containing the sequence | arrangement in any one of sequence number 9-12;
8). A transcription unit comprising the DNA according to 6 or 7 above, comprising a promoter in a form capable of controlling the DNA on the 5 ′ side, and a terminator on the 3 ′ side of the DNA;
9. 9. The transcription unit according to 8 above, wherein the promoter is selected from the group consisting of a tRNA promoter, an H1 promoter, a U6 promoter, a polymerase II promoter and a CMV promoter;
10. 10. The transcription unit according to 8 or 9 above, wherein the terminator is a terminator comprising a sequence in which at least 4 or more thymidine residues are continuous;
11. A vector comprising the transcription unit according to any one of 8 to 10 above;
12 The vector according to 11 above, wherein the vector can be introduced into a mammal;
13. At least selected from the group consisting of the RNA according to any one of 1 to 5, the DNA according to 6 or 7, the transcription unit according to any of 8 to 10 and the vector according to 11 or 12. A composition for treating a cell proliferative disease, comprising a composition comprising one or more, wherein the composition suppresses the expression of TS in a human cell to which the composition is administered;
14 14. The composition according to the above 13, wherein the cell proliferative disease is cancer, and cell proliferation is suppressed by suppressing the expression of TS;
About.

  The present invention provides an RNAi molecule that efficiently suppresses the expression of TS and a DNA encoding the RNAi molecule. The composition containing the RNAi molecule or the DNA molecule is an effective novel therapeutic agent regardless of the type of cancer, and administration of such a therapeutic agent is a therapeutic method with extremely reduced side effects.

  In the first aspect, the present invention provides a double-stranded RNA that can act as a human TS-specific RNAi molecule. It is a double-stranded RNA that acts as an RNAi molecule, the sequence of which includes a strand that is complementary to the sequence of the target mRNA. Therefore, in order to design an RNAi molecule for suppressing human TS expression, the present inventors select as many as 12 kinds of candidate sequences according to a conventional method, and detect the action effect of the RNAi molecule having the sequence. Thus, it was found that only those having a specific sequence exhibited a significant effect, and the present invention was completed. Methods for selecting RNAi molecule sequences are described in various documents. For example, SM Elbashir et al. (2001) Nature 411: 494-498, SM Elbashir et al. (2001) Genes & Dev. 15: 188-200, SM Elbashir et al. (2001). EMBO J. 20: 6877-6888, Q. Ge (2003) Proc. Natl. Acad. Sci. USA 100: 2718-23, J. Harborth et al. (2001). J. Cell Science 114: 4557-4565, J. Harborth et al. (2003) Antisense Nucleic Acid Drug Dev. 13: 83-106, JR Masters et al. (2001) Proc. Natl. Acad. Sci. USA 98: 8012-8017, T. Tuschl et al. (1999) Genes & Dev. 13: 3191-3197. However, RNAi molecules having sequences selected according to the description do not necessarily show the effects of RNAi as expected, but it is only possible to confirm the effects in practice, and the design of RNAi molecules with effects is the current technology In terms of standards, it is a difficult task. As a result of searching for the sequence of a TS-specific RNAi molecule and overcoming this problem, the present inventors have found that the RNA strand acting as an RNAi molecule for human TS is complementary to the sequence described in SEQ ID NOs: 1-4. It was deduced that each was a double-stranded RNA consisting of a simple sequence.

  However, even a sequence containing a single base deletion, substitution or addition in the sequence may act as an RNAi molecule.

  Furthermore, since it is desirable that the RNA molecule that can act as RNAi has a length of 19 bases or more and 30 bases or less, from at least 19 consecutive bases of the sequence consisting of 30 bases shown in SEQ ID NOs: 1-4. RNA comprising the sequence However, it is preferable that the length does not exceed 30 bases.

  Further, since the RNAi molecule is a double-stranded RNA, it is necessary to form a double strand by hybridizing the RNA having the above sequence and the RNA hybridized therewith. Here, the sequences that hybridize with any one of SEQ ID NOs: 1 to 4 are preferably completely complementary to each other, but may be sequences that hybridize under stringent conditions. The stringent condition refers to, for example, a condition where the sodium concentration is 10 mM to 300 mM, preferably 42 to 55 ° C, more preferably 42 ° C. Alternatively, it may be a sequence having 90% or more, preferably 93% or more, more preferably 96% or more identity with a sequence completely complementary to any one of SEQ ID NOs: 1 to 4.

  A double-stranded RNA (dsRNA) molecule that acts as a preferred RNAi molecule is a hybrid between a single-stranded RNA having the sequence shown in any of SEQ ID NOs: 1 to 4 and a single-stranded RNA having a completely complementary sequence thereto. Provided in soy form.

  The RNAi molecule may be provided as a hairpin dsRNA in which a single RNA strand forms a hairpin structure. In the present specification, the RNAi molecule refers to an RNA molecule that exhibits an RNA interference action, and includes both shRNA (short hairpin RNA) and siRNA (small interfering RNA). In such a hairpin dsRNA, the loop portion of the hairpin is single-stranded, but the loop portion is cleaved when acting as an RNAi molecule. The sequence of the loop part of the hairpin dsRNA is preferably a sequence selected from the group consisting of CUUCCUGUCA (SEQ ID NO: 27), UUCAAGAGA, CCACC, CUCGAG, CCACACC, UUCAAGAGA, AUG, CCC and UUCG. That is, CUUCCUGUCA (SEQ ID NO: 27), UUCAAGAGA, CCACC, CUCGAG, CCACACC, UUCAAGAGA, AUG, CCC are adjacent to the 3 ′ end of any one of the sequences shown in SEQ ID NOs: 1-4. And a sequence selected from the group consisting of UUCG, and adjacent to the 3 ′ end thereof, complementary or stringent conditions to the selected one of the sequences shown in SEQ ID NOs: 1 to 4 Single-stranded RNA molecules having sequences to which sequences that hybridize below are linked are preferred as hairpin dsRNAs. For example, the sequence shown to sequence number 5-8 is mentioned.

  These RNA molecules can be injected into an individual where cell growth inhibition is desired, or delivered to a local area where it is desired to inhibit cell growth, such as within a malignant tumor cell. Suppresses the expression of TS and suppresses or blocks cell proliferation. The injection method to the individual can be performed by a known method such as injection. Since RNA molecules can undergo metabolic degradation in blood, it is desirable to modify them by known nucleic acid modification methods to prevent this. The amount to be injected depends on the stability of the RNA molecule, the efficiency of local delivery, and the like. As a method for local delivery, it can be directly injected into a target region, but various techniques such as liposomes and polymer micelles have been developed, and these may be used appropriately.

  The dsRNA may be synthesized in vivo. That is, DNA encoding the above RNA can be introduced into a cell and transcribed in vivo by the cell's endogenous RNA polymerase to generate dsRNA. Therefore, DNA encoding the above RNA can also be included in the scope of the present invention. For example, DNA which has the sequence shown to sequence number 9-12 which codes the RNA which has the sequence shown to sequence number 5-8 which is the said hairpin type RNAi molecule | numerator (shRNA) is mentioned. Transcription of the DNA is preferably controlled by regulatory regions such as promoters, enhancers, silencers, splicing donors and acceptors, and poly A, and in particular, the promoter is present at the 5 ′ end of the DNA, and the promoter It is preferred that the transcription of DNA is controlled. Examples of particularly preferred promoters include, but are not limited to, tRNA promoters, H1 promoters, U6 promoters, polymerase II promoters, and CMV promoters. A terminator for terminating the transcription of the DNA is preferably linked to the 3 'side of the DNA. Preferred terminators include, but are not limited to, a terminator consisting of a sequence of at least four or more thymidine residues, particularly Tx7. Regulatory regions, promoters and terminators other than those exemplified herein can also be useful in the present invention, and selection of appropriate ones can be easily performed by those skilled in the art. A transcription unit comprising DNA encoding the RNA, a promoter on the 5 'side so that the DNA can be controlled, and a terminator on the 3' side of the DNA is also included in the scope of the present invention.

  Furthermore, the transcription unit of the present invention can be inserted into a vector, the vector can be introduced into a target individual or a cell of an individual tissue, and can act as an RNAi molecule by transcription from the transcription unit in the vector. It is possible to generate RNA molecules. The vector may be a plasmid vector or a viral vector, and is preferably one that can be introduced into a human or mouse target cell and can be amplified in the cell. For example, when the plasmid piGENE ™ tRNAPur is used, the vector is designed to include a tRNA promoter, a Tx7 terminator, and a plurality of restriction enzyme sites for cloning between them. The transcription unit described above is designed by synthesizing and synthesizing a DNA in which the coding restriction DNA site is linked to a linker so that the restriction endonuclease site matches the cleavage end and its complementary strand, and inserting it into the vector in a double-stranded form. Can be constructed.

  Examples of methods for introducing such vectors into predetermined cells in the living body include lipid-mediated carrier transport methods (eg, lipofectamine method), chemical substances (calcium phosphate, etc.)-Mediated transport, microinjection, genes There are a gun driving method, an electroporation method, and the like. Those skilled in the art can select an appropriate method from known methods.

  Vectors containing DNA encoding RNAi molecules are less expensive than synthesizing double-stranded RNA or loop RNA, and can be amplified after introduction into cells to stably produce large amounts of RNAi molecules. Therefore, it is more advantageous to transfect cells with such vectors than to introduce double-stranded RNA or loop RNA into cells, because it is more quantitatively efficient than RNA. Conceivable. In addition, in order to introduce double-stranded RNA or loop-type RNA into cells, double-stranded RNA or loop-type RNA is obtained by in vitro transcription or in vivo transcription using a vector containing the DNA encoding them. It can also be manufactured.

  As described above, the RNA or DNA capable of generating the RNAi molecule for human TS of the present invention acts as an RNAi molecule in the cell, suppresses the expression of TS in the cell, and suppresses cell proliferation. It is possible to treat a disease, particularly a malignant tumor including cancer, or to reduce the symptoms of the disease and to suppress progression. In the present specification, the “cell proliferative disease” is a disease in which abnormal cell growth is observed, and includes, for example, various cancers (malignant tumor, lymphoma, etc.).

  That is, the pharmaceutical composition for suppressing the expression of TS containing the RNAi molecule of the present invention (including double-stranded RNA and hairpin RNA) or DNA encoding the same as an active ingredient is also within the scope of the present invention. Is within. The composition may contain a pharmaceutically acceptable carrier and may contain various useful additives such as a stabilizer for enhancing the stability of the active ingredient. The DNA may be present in the transcription unit or in the vector as described above. The pharmaceutical composition is useful for the treatment or prevention of human cell proliferative diseases, particularly malignant tumors.

  A method of treating a cell proliferative disease, particularly a malignant tumor, comprising administering the RNAi molecule of the present invention or a DNA encoding the same can also be included in the scope of the present invention.

1. Construction of DNA Vector Encoding RNAi Molecule (shRNA) for Human TS First, DNA having the sequences shown in SEQ ID NOs: 13 to 18 was synthesized by a conventional method. The DNA of the sequence shown in SEQ ID NO: 18 encodes an RNAi molecule for TGFβ receptor 2, but was used as a negative control. All of the sequences shown in SEQ ID NOs: 13 to 17 are DNAs encoding RNA homologous to a part of human TS mRNA (sense strands: SEQ ID NOs: 1 to 4 and 19) and RNAs complementary to the sequences (anti-antigens). A DNA encoding a sense strand) and a DNA encoding an RNA (loop portion) having the sequence shown in SEQ ID NO: 27 between them. The sequence shown in SEQ ID NO: 18 contains RNA homologous to a part of human TGFβ receptor 2 mRNA (sense strand: SEQ ID NO: 20) instead of the RNA homologous to a part of human TS mRNA. Further, for insertion into the piGENETM tRNA vector, there is a SacI site linking linker on the 5 ′ end side and a KpnI site linking linker on the 3 ′ end side. Furthermore, the other DNA strands for each of these are synthesized, which are complementary to each other except for the linker site, and have a linker for linking the KpnI site on the 5 ′ end side and a linker for linking the SacI site on the 3 ′ end side (sequence) Numbers 21-26). When the two synthesized DNA strands (see FIG. 1) are annealed, there are protruding ends that can be ligated with SacI and KpnI cleavage sites at both ends. In this way, the two synthesized DNAs are annealed and ligated to the site of the piGENE ^™ tRNA vector treated with SacI and KpnI to obtain piGENETM tRNA containing human TSshRNA-encoded DNA.
Respectively, and ^{Sh TS 115, Sh TS 141,} Sh TS 269, Sh TS 289, Sh TS 202 and Sh TR2 ^130.

2. Confirmation of RNAi action using human hepatoma cell line This is transfected into Escherichia coli and a plasmid is prepared. All of the above can be performed according to a conventional method.

The obtained plasmid is transfected into a human intrahepatic cholangiocarcinoma-derived HuCC-T1 cell line as follows. The cell line is usually cultured and maintained in RPMI 1640 containing 10% fetal calf serum (FCS) (with 100 U / mL penicillin and 100 μg / mL streptomycin) at 37 ° C. in a 5% CO ₂ environment. The cells were seeded in a 48-well plate at a density of 5 × 10 ⁴ cells / well. After 24 hours, when the cells were 70-90% confluent, 0.6 μg of the above plasmid DNA was dissolved in 50 μl of Opti-MEM I Reduced-Serum Medium (Gibco) and 1 μl of lipofectamin2000 was dissolved in 50 μl of OPTIMEM I. Those dissolved in the medium are allowed to stand at room temperature for 5 minutes after being dissolved, and then mixed and incubated at room temperature for 20 minutes. This is added to a well containing the above cells whose medium has been changed with 300 μl / well of OPTIMEM 1, and cultured at 37 ° C. After 4 hours, change the medium and return to normal RPMI1640 medium.

72 hours after the transfection, RNA was extracted and purified from cells using isogen (Nippon Gene), and the amount of TS mRNA was quantified by real-time PCR.
As shown in FIG. 2, among the above vectors, the cells transfected with Sh TS ²⁸⁹ , Sh TS ²⁶⁹ , Sh TS ¹⁴¹ , Sh TS ¹¹⁵ showed a significant decrease in the amount of TS mRNA compared to the control (Sh TS ^NC ). However, in the cells transfected with Sh TR2 ¹³⁰ and Sh TS ²⁰² , the amount of TS mRNA tended to increase. At this time, the amount of rRNA was used as an internal control. Next, Western blot was performed to confirm the expression level at the protein level. In the Western blot, the amount was compared with GAPDH as the internal control.Similar to the results of real-time PCR mRNA amount, only those transfected with Sh TS ²⁸⁹ , Sh TS ²⁶⁹ , Sh TS ¹⁴¹ , Sh TS ¹¹⁵ were used as controls. In comparison, the amount of protein was reduced (FIG. 3).

  From the above examples, it can be seen that the expression of TS can be suppressed by the RNAi molecule of the present invention, whereby cell proliferation can be suppressed.

The DNA (TSHs115, TSHs141, TSHs269, TSHs289) which codes the human TSshRNA which connected the linker for inserting in piGENETM tRNA vector is shown. It consists of a sense RNA coding part consisting of 30 residues, a loop part and an antisense RNA coding part, and has restriction enzyme sites at both ends. It shows the TSmRNA levels of ^{Sh TS 269, Sh TS 141,} Sh TS 115, Sh TS 289, Sh TS 202 and cells and negative control for the sh TR ¹³⁰ was transfected (Sh TS ^NC) was transfected cells. Negative controls are cells transfected with an empty piGENE ^™ tRNA vector. Sh TR ¹³⁰ was also used as a negative control. Shows TS protein levels in cells transfected with plasmids containing Sh TS ²⁶⁹ , Sh TS ¹⁴¹ , Sh TS ¹¹⁵ , Sh TS ²⁸⁹ , Sh TS ²⁰² and EGFP coding sequence and cells transfected with negative control (Sh TS ^NC ) . Negative controls are cells transfected with an empty piGENE ^™ tRNA vector. An EGFP expression plasmid was also used as a negative control.

Claims (12)

The following (a) and (b):
(A) RNA comprising the sequence shown in any one of SEQ ID NOs: 1 to 4;
(B) completely complementary sequence in any of SEQ ID NO: 1-4 or Ranaru RNA;
Is a double-stranded RNA hybridized, which can act as an RNAi molecule for targeting human thymidylate synthase (TS) and reducing the expression level of the enzyme in cells. The following (a) and (b):
(A) RNA comprising the sequence shown in any one of SEQ ID NOs: 1 to 4;
(B) completely complementary sequence in any of SEQ ID NO: 1-4 or Ranaru RNA;
Are connected in tandem, and the connected part is
(C) RNA having one sequence selected from the group consisting of CUUCCUGUCA (SEQ ID NO: 27), UUCAAGAGA, CCACC, CUCGAG, CCACACC, UUCAAGAGA, AUG, CCC and UUCG;
(C) is an RNA characterized in that the portion (c) takes a loop structure when the above (a) and (b) hybridize to form a double strand. RNA that can act as an RNAi molecule for targeting human thymidylate synthase (TS) and reducing the expression level of the enzyme in cells.   The RNA according to claim 2, comprising the sequence shown in any one of SEQ ID NOs: 5 to 8. DNA encoding the RNA according to any one of claims 1 to 3 . The DNA according to claim 4 , comprising the sequence shown in any one of SEQ ID NOs: 9 to 12. A transcription unit comprising the DNA according to claim 4 or 5 , comprising a promoter on the 5 'side so that the DNA can be controlled, and a terminator on the 3' side of the DNA. The transcription unit according to claim 6 , wherein the promoter is selected from the group consisting of a tRNA promoter, an H1 promoter, a U6 promoter, a polymerase II promoter and a CMV promoter. The transcription unit according to claim 6 or 7 , wherein the terminator is a terminator comprising a sequence in which at least four or more thymidine residues are continuous. A vector comprising the transcription unit according to any one of claims 6 to 8 . The vector according to claim 9 , wherein the vector can be introduced into a mammal. The RNA according to any one of claims 1 to 3 , the DNA according to claim 4 or 5 , the transcription unit according to any one of claims 6 to 8 , and the vector according to claim 9 or 10. A composition for treating a cell proliferative disease, comprising a composition comprising at least one selected from the group consisting of: inhibiting TS expression in human cells to which the composition is administered. The composition according to claim 11 , wherein the cell proliferative disease is cancer, and cell proliferation is suppressed by suppressing expression of TS.

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